Nonulosonic sugars are a family of nine-carbon α-keto acids that are predominantly found on the outer surfaces of both eukaryotic and bacterial cells. Sialic acid (see FIG. 1), which is the best characterized member of this family, plays a crucial role in animal physiology; for this reason, sialic acid and its derivatives have been extensively used as drug targeting molecules (Javant et al, 2007), anti-viral drugs (von Itzstein, 2007), cell-imaging agents (Mahal et al, 1997; Hsu et al, 2007), and as supplements in nutraceuticals (Colombo et al, 2003; Wang et al, 2007).
Two other structurally distinct nonulosonic sugars, pseudaminic (Schoenhofen et al, 2006a) (Pse) and legionaminic (Schoenhofen et al, 2009) (Leg) acid (FIG. 1), and their biosynthetic pathways have also been characterized. These sialic acid-like sugars are constituents of microbial glycans, which are associated with important virulence factors, including flagella (McNally et al, 2007), capsules (Kiss et al, 2001), and lipopolysacchrides (Knirel et al, 2003 (LPS). Many commensal, as well as pathogenic bacteria, notably Campylobacter jejuni, Campylobacter coli, Clostridium botulinum, Escherichia coli O161, Helicobacter pylori, Legionella pneumophila, Vibrio parahaemolyticus and Pseudomonas aeruginosa, biosynthesize and decorate their surfaces with these nonulosonic acids, whose function(s) remains unclear.
The structural similarities and evolutionary history (Lewis et al, 2009) shared between these three nonulosonic sugars has raised considerable interest in understanding the mammalian sialobiology associated with bacterial-derived Pse and Leg. The biosynthesis of Pse and Leg parallels that of sialic acid and involves the condensation of a 6-carbon amino sugar intermediate with phosphoenolpyruvate (PEP) to generate the corresponding nonulosonate. In contrast to sialic acid biosynthesis in bacteria, which involves condensation of N-acetylmannosamine (ManNAc) with PEP, both Pse and Leg utilize unusual 2,4-diacetamido-2,4,6-trideoxy hexoses (DATDH) for the synthase step.
Pse is biosynthesized from UDP-N-acetylglucosamine (UDP-GlcNAc) in a five-step enzymatic transformation (Schoenhofen et al, 2006a) (see Table 1 and FIG. 2). A dedicated dehydratase (PseB) and aminotransferase (PseC) pair (Schoenhofen et al, 2006b), converts UDP-GlcNAc into UDP-4-amino-4,6-dideoxy-β-L-AltNAc. An acetyltransferase, PseH, and a UDP-sugar hydrolase, PseG, transform this UDP-activated sugar intermediate into 2,4-diacetamido-2,4,6-trideoxy-L-altropyranose (6-deoxy-AltdiNAc). The Pse synthase, PseI, performs the PEP-dependent condensation with 6-deoxy-AltdiNAc to liberate 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid or pseudaminic acid.
TABLE 1The enzymes involved in UDP-6-deoxy-AltdiNAc, pseudaminic acid (Pse),UDP-BacdiNAc and legionaminic acid (Leg) biosynthesis. Enzymes areshown in sequential order, where each product is a substrate for the nextbiosynthetic step. The initial substrate for each pathway is UDP-GlcNAc.EnzymeIn vitro EnzymeNomenclaturefunctionBiosynthetic product(s)UDP-6-deoxy-AltdiNAc and Pseudaminic acid routePseBUDP-GlcNAc 4,6-UDP-2-acetamido-2,6-dideoxy-β-L-arabino-hexos-dehydratase and 5-4-uloseepimerasePseCaminotransferaseUDP-4-amino-4,6-dideoxy-β-L-AltNAc orUDP-2-acetamido-4-amino-2,4,6-trideoxy-β-L-AltPseHN-acetyltransferaseUDP-2,4-diacetamido-2,4,6-trideoxy-β-L-Alt(UDP-6-deoxy-AltdiNAc)PseGUDP-sugar hydrolase2,4-diacetamido-2,4,6-trideoxy-L-Alt(6-deoxy-AltdiNAc)PseIPse synthase5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse)UDP-BacdiNAc and Legionaminic acid routePglFUDP-GlcNAc 4,6-UDP-2-acetamido-2,6-dideoxy-α-D-xylo-hexos-4-dehydrataseulosePglEaminotransferaseUDP-4-amino-4,6-dideoxy-α-D-GlcNAc orUDP-2-acetamido-4-amino-2,4,6-trideoxy-α-D-GlcPglDN-acetyltransferaseUDP-2,4-diacetamido-2,4,6-trideoxy-α-D-Glc(UDP-BacdiNAc)LegGUDP-sugar hydrolase2,4-diacetamido-2,4,6-trideoxy-D-Manand 2-epimerase(6-deoxy-MandiNAc)LegILeg synthase5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid (Leg)
Leg may also be biosynthesized from UDP-N-acetylglucosamine (UDP-GlcNAc) in a five-step enzymatic transformation (Schoenhofen et al, 2009; Schoenhofen et al, 2006b; Oliver et al, 2006; Glaze et al, 2008; see Table 1 and FIG. 2). Dehydratase PglF and aminotransferase PglE pair (Schoenhofen et al, 2006b) convert UDP-GlcNAc into UDP-4-amino-4,6-dideoxy-α-D-GlcNAc. Acetyltransferase PglD and hydrolyzing 2-epimerase LegG transform this UDP-activated sugar intermediate into 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose (6-deoxy-MandiNAc). The Leg synthase, LegI, performs the PEP-dependent condensation with 6-deoxy-MandiNAc to liberate 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid (legionaminic acid). Enzymes functionally similar to PglF, PglE and PglD also exist, for example the C. jejuni LegB, LegC and LegH (respectively) enzymes, which produce identical sugar products except that they are GDP-linked biosynthetic intermediates (Schoenhofen et al, 2009). Here, PglFED produce UDP-BacdiNAc starting from UDP-GlcNAc (see Table 1). Similarly, certain LegG enzymes, as in C. jejuni, may utilize a GDP-linked substrate
The bacterial nonulosonic acids are a medically and biotechnologically important family of cell-surface carbohydrates. Current methods for producing these complex sugars allows the isolation of only limited, sub-gram quantities from natural resources, or via currently available chemical or enzymatic synthesis in vitro. Additionally, the cost of enzyme preparation, reagents and cofactors required for in vitro synthesis is quite significant.
There remains a need in the art for a method that can generate significant quantities of Pse and Leg in a cost-effective manner.